PROJECT SUMMARY/ABSTRACT This project is a combined design-driven and hypothesis-driven project to bioengineer microscale models of enteric disease. Starting with the Spence lab's in vitro intestine system that accurately reflects both the complex cellular makeup and the appropriate layered organization of the human intestine, this project will provide these 3-Dimensional (3D) Human Intestinal Organoids (HIOs) with physiologicaly soft but confining mechanical cues as well as microscale fluid perfusion capabilities that will mimic luminal flow, to further induce physiological structures such as crypts and villi. Both of these properties (constraint, flow) have a significant impact on intestine development, differentiation and function. Our hypothesis is that by providing a mechanically confined culture condition and fluid perfusion, as opposed to the free expanding culture with a static, enclosed lumen as is currently used for HIO formation, that the epithelial layer will self-organize additional levels of physiological complexity, including as crypts and villi, along with associated spatial organization of intestinal stem cells (ISCs) in crypts and differentiated cells on the villi. Incorporation of microscale fluid perfusion capabilities in HIO culture devices will also allow precise regulation of intraluminal flow of nutrients, and long-term colonization with bacteria, and pathogens. Technologically, this project will be innovative in developing a method (?supersoft lithography?) for reproducibly creating supersoft PDMS structures with physiological moduli of 1-100 kPa. To enable closed-loop control for maintenance of tissue homeostasis as well as to provide readouts of tissue function, this project will also integrate miniature oxygen sensors and electrodes for trans-epithelial electrical resistance (TEER) measurements. Additionally, sampling capabilities from the interior and exterior of the HIO will be incorporated to enable off-line measures of fluid and drug absorption/secretion. HIO microscale culture devices will also facilitate measurement of cytokine production in integrated HIO-immune co-cultures. Finally, we will demonstrate modularity and utility of the bioengineered and instrumented HIO system by integrating NAMSED Projects 1, 2 and 3. Specifically, instrumented-HIOs with luminal flow will be generated, co-cultured with immune cells and colonized by probiotic microbes (Lactobacillus GG, LGG) and/or pathogens (S.typhimurium). In each co-culture, (probiotic/HIO/immune vs. probiotic/pathogen/HIO/immune), we will test the ability of the system to generate real-time physiological data by measuring epithelial barrier function (TEER, FITC-Dextran), oxygen concentration, cytokine production, and finally by examining epithelial invasion by S.typhimurium. We will also test the utility of this system to screen drugs/compounds by generating instrumented LGG/S.typhimurium/HIO/immune co-cultures and adding Cefoperazone, an antibiotic that will selectively target the pathogen S.typhimurium, but not the probiotic LGG. The ability of Cefoperazone to kill S.typhimurium will be examined by culturing the luminal effluent to determine S.typhimurium colony forming units before, during and after antibiotic treatment. Finally, when live cultures are terminated, we will harvest the system and examine cellular and molecular difference between the different groups using immunofluorescence or qRT-PCR on purified immune cells and epithelium.